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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 11 (December 1), 1998:
pp. 4220-4229
Proteasome Inhibitors Induce Apoptosis in Glucocorticoid-Resistant
Chronic Lymphocytic Leukemic Lymphocytes
By
Joya Chandra,
Irina Niemer,
Joyce Gilbreath,
Kay-Oliver Kliche,
Michael Andreeff,
Emil J. Freireich,
Michael Keating, and
David J. McConkey
From the Departments of Cell Biology and Hematology, The University
of Texas, M.D. Anderson Cancer Center, Houston, TX.
 |
ABSTRACT |
Our previous work showed that the nuclear scaffold (NS) protease is
required for apoptosis of both thymocytes and chronic lymphocytic
leukemic (CLL) lymphocytes. Because partial sequencing of one of the
subunits of the NS protease revealed homology to the proteasome, we
tested the effects of classical proteasome inhibitors on apoptosis in
CLL cells. Here we report that proteasome inhibition caused high levels
of DNA fragmentation in all patients analyzed, including those
resistant to glucocorticoids or nucleoside analogs, in vitro.
Proteasome inhibitor-induced DNA fragmentation was associated with
activation of caspase/ICE family cysteine protease(s) and
was blocked by the caspase antagonist, zVADfmk. Analysis
of the biochemical mechanisms involved showed that proteasome inhibition resulted in mitochondrial dysregulation leading to the
release of cytochrome c and a drop in mitochondrial transmembrane potential (  ). These changes were associated with inhibition of
NF B, a proteasome-regulated transcription factor that has been
implicated in the suppression of apoptosis in other systems. Together,
our results suggest that drugs that target the proteasome might be
capable of bypassing resistance to conventional chemotherapy in CLL.
© 1998 by The American Society of Hematology.
| |
INTRODUCTION |
CHRONIC LYMPHOCYTIC leukemia (CLL) is an
illness characterized by an accumulation of monoclonal mature B cells
in the peripheral blood. Although CLL is the most common leukemia in the Western world, little is known about the biology of the disease. Treatment schemes rely heavily on glucocorticoids, chlorambucil, and
nucleoside analogs, and we and others have shown that all of these
agents trigger apoptosis in CLL cells in vitro, suggesting that
induction of apoptosis may account for their therapeutic efficacy.
Furthermore, recent work has shown that apoptosis in vitro correlates
with Rai stage,1,2 and rates of apoptosis detected
following fludarabine treatment correlate with clinical response in
vivo.3 However, despite the initial effectiveness of these
drugs in patients with low-grade disease, resistant cells ultimately
emerge, leaving no effective treatment options available. It is
possible that drug-resistant CLL cells possess intrinsic defect(s) in
their ability to undergo apoptosis.
Protease activation is required for completion of the apoptotic program
in all cellular and cell-free systems interrogated to
date.4,5 Of central importance are members of the
ICE/caspase family of aspartate-specific cysteine proteases, which
appear to function at the core of the "effector" machinery for
cell death. Caspase activation can either be directly promoted by
oligomerization of certain caspase-associated cell surface
"death" receptors (Fas, TNF-RI)6 or by
intramitochondrial events that lead to the release of the electron
transport chain intermediate, cytochrome c.7 Precisely how
caspases promote the downstream features of apoptosis is not clear, but
studies with specific peptide-based active site inhibitors indicate
that they are required for all of the major biochemical events observed
in apoptotic cells, including changes in cellular morphology, loss of
plasma membrane asymmetry (exposure of phosphatidylserine on the outer
leaflet), and DNA fragmentation.8
We and others have obtained evidence that certain noncaspase proteases
are also required for DNA fragmentation and apoptosis. Specifically, we
have shown that peptide-based active site inhibitors of a
Ca2+-dependent nuclear protease, termed the nuclear
scaffold (NS) protease, block glucocorticoid- and nucleoside
analog-induced DNA fragmentation in CLL lymphocytes.9
Although the molecular characteristics of the NS protease are at
present unclear, preliminary evidence obtained by another
laboratory10 suggests that it is structurally and
functionally related to the 26S multicatalytic protease complex (MPC),
otherwise known as the proteasome. This possible similarity may explain
why NS protease inhibitors block DNA fragmentation, because previous
studies have implicated the proteasome in the programmed cell death of
intersegmental muscles in the moth, Manduca
sexta,11 and more recent work in isolated mouse
thymocytes12 and neuronal cells13 has
shown that proteasome inhibitors block caspase activation and other
downstream events associated with apoptosis in these cells.
The results presented above suggested to us that the effects of NS
protease inhibitors in CLL cells might be due to proteasome inhibition.
To directly address this possibility, we tested the effects of several
specific proteasome inhibitors on caspase activation and DNA
fragmentation in isolated CLL lymphocytes, expecting that they would
suppress apoptotic cell death. On the contrary, here we report that
proteasome inhibition resulted in extraordinarily high levels of DNA
fragmentation in all patient isolates analyzed, including those found
to be completely resistant to glucocorticoid-induced apoptosis.
Analysis of the biochemical mechanisms involved showed that the effects
are linked to inhibition of NFB, a transcription factor implicated
in the maintenance of cell survival in other model
systems.14-17
| |
MATERIALS AND METHODS |
Materials.
The esterified peptide caspase inhibitor, Z-VAD (OMe)fmk, the
fluorigenic caspase substrate, DEVD-AMC, and the mouse anti-PARP monoclonal antibody C2-10 were purchased from Enzyme Systems Products, Inc (Dublin, CA). A peptide inhibitor of the NS protease, Z-APFcmk, and
the caspase antagonist, Boc-Asp-chloromethylketone (BDcmk) were
purchased from Bachem Bioscience (King of Prussia, PA). Monoclonal antibodies for caspase-3, p53, p27, and c-Jun were purchased from Transduction Laboratories (Lexington, KY). A monoclonal antibody to
c-Fos and the proteasome inhibitors lactacystin and MG-132 were
obtained from Calbiochem (San Diego, CA). Horseradish
peroxidase-conjugated anti-mouse and anti-rabbit antibodies were from
Amersham Corp (Arlington Heights, IL).
Patients, cell isolation, and incubation criteria.
All patients fulfilled the National Cancer Institute's (NCI) criteria
for the diagnosis of CLL. Some of the patients had received prior
therapy, although none within the last 6 months before experimentation. Immunophenotyping by dual-parameter flow cytometry showed coexpression of CD5 with B-cell antigen and isotypic light chain expression. Clinical staging was based on the system described by
Rai.18 Freshly isolated peripheral blood was fractionated
by Ficoll-Hypaque (Winthrop Pharmaceuticals, New York, NY)
sedimentation at 4°C. Nonadherent mononuclear cells were then
immediately suspended in complete RPMI 1640 medium supplemented with
10% fetal calf serum (FCS), 10 mmol/L HEPES (pH 7.5), and antibiotics
at a cellular concentration of 1 to 2 × 106 cells/mL.
Cell viability was assessed by Trypan blue exclusion and exceeded 95%
following the isolation procedure.
Granulocyte-colony stimulating factor (G-CSF)-mobilized progenitor
cells were obtained from pheresis samples by magnetic cell sorting
(MACS). The pheresis samples were resuspended in 50 mL of cold RPMI
medium, and two "soft-spins" (200g, 10 minutes) were performed to remove platelets. Cells were labeled with anti-CD34 antibody and isolated with a commercial CD34 isolation kit (Miltenyi Biotec, Auburn, CA) according to the manufacturer's instructions. A
MACS buffer consisting of Ca2+/Mg2+-free
Hanks' buffered salt solution (HBSS) containing 0.6% ACD-A (Baxter,
Deerfield, IL), 0.5% bovine serum albumin (BSA; Sigma, St Louis, MO),
pH 6.5, was used throughout staining and separation to prevent cell
clumping while maintaining optimal progenitor viability. Cells were
separated on VS-positive selection columns using a VarioMACS according
to the manufacturer's instructions. Cell purity was assessed by flow
cytometry using CD34-phycoerythrin (PE) and CD45-fluorescein
isothiocyanate (FITC) as described previously.19
DNA fragmentation analysis.
Quantification of apoptosis by propidium iodide (PI) staining and
fluorescence-activated cell sorting (FACS) analysis was performed as
described previously.20 Following incubation with various
agents in vitro, cells were pelleted by centrifugation and resuspended
in phosphate-buffered saline (PBS) containing 50 µg/mL PI, 0.1%
Triton X-100, and 0.1% sodium citrate. Samples were stored at 4°C
for 16 hours and vortexed before FACS analysis (FL-3 channel).
Cytochrome c release measurements.
Release of cytochrome c from mitochondria was measured by
immunoblotting essentially as described previously.21 Cells
were incubated in the absence or presence of 10 µmol/L APFcmk, 10 µmol/L MG-132, or 10 µmol/L methylprednisolone for 4 hours,
obtained by centrifugation, and gently lysed for 30 seconds in an
ice-cold buffer containing 250 mmol/L sucrose, 1 mmol/L EDTA, 0.1%
digitonin, and 25 mmol/L Tris, pH 6.8. Lysates were centrifuged for 2 minutes at 12,000g, supernatants were mixed with 2×
Laemmli's reducing sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) sample buffer, and extracts from equal
numbers of cells (10 to 20 × 106) were resolved by
15% SDS-PAGE. Polypeptides were transferred to nitrocellulose
membranes (0.2 µm; Schleicher & Scheull, Keene, NH), and cytochrome c
was detected by immunoblotting with a monoclonal antibody (clone
7H8.2C12; purchased from Pharmingen, San Diego, CA).
Caspase activity assay.
Protease activity measurements were conducted as described
previously.9 Cells were lysed in 1 mL of a buffer
containing 25 mmol/L HEPES (pH 7.4), 5 mmol/L EDTA, 2 mmol/L
dithiothreitol, and 10 µmol/L digitonin for 15 minutes on ice. The
lysates were clarified by centrifugation (12,000g), and
supernatants were incubated with 50 µmol/L
Asp-Glu-Val-Asp-aminomethylcoumarin (DEVD-AMC; Enzyme Systems Products,
Inc at 37°C in the dark. Relative activities were then measured in
a spectrofluorimeter (400 nm excitation, 505 nm emission); blanks
included supernatants processed as outlined above without dye and
supernatants preincubated with BDcmk (25 µmol/L).
Mitochondrial membrane potential measurements.
The potential-sensitive fluorochrome JC-1 (Molecular Probes, Eugene,
OR) was used to measure  mito. Cells were obtained by centrifugation and incubated with 10 µmol/L JC-1 for 15 minutes at
37°C in the dark. Cells were washed in PBS and analyzed by FACS on
the FL-2 channel (FACScan; Becton Dickinson, Mountain View, CA).
Annexin V binding.
Exposure of surface phosphatidylserine was quantified by surface
annexin V staining as described previously.22 This assay was used as a DNA fragmentation-independent endpoint to confirm the
involvement of apoptosis in the mechanism of cell death. Cells were
resuspended in binding buffer containing 1 µg/mL FITC-conjugated annexin V (Nexins Research B.V., Hoeven, The Netherlands) and incubated
for 30 minutes at 4°C, and cells were analyzed by flow cytometry
(FACScan, Becton Dickinson).
Immunoblotting.
For detection of caspase-3, p53, p27, Fos, and Jun, cells were lysed
for 1 hour at 4°C in a buffer containing 150 mmol/L NaCl, 1%
Triton X-100, a cocktail of protease inhibitors (Complete Mini tablets;
Boehringer-Mannheim, Indianapolis, IN), and 25 mmol/L Tris
(pH 7.5). Debris was sedimented by centrifugation for 5 minutes at
12,000g, and the supernatants were solubilized for 5 minutes at
100°C in Laemmli's SDS-PAGE sample buffer containing 100 mmol/L dithiothreitol.
For analysis of PARP cleavage samples were denatured in a urea/SDS
buffer as follows: cells were incubated with various agents for 16 hours, obtained by centrifugation, and resuspended in 25 µL ice-cold
PBS. Cells were subsequently disrupted by addition of 100 µL of a
buffer containing 6 mol/L urea, 2% SDS, 10% glycerol, 5 mmol/L EDTA,
5% 2-mercaptoethanol, and 100 mmol/L Tris (pH 6.8) followed by
pipeting through a 1-mL Pipetman tip, and samples were sonicated for 20 seconds at high power to sheer DNA. Samples were then incubated for 15 minutes at 65°C and centrifuged for 2 minutes at 12,000g
before they were loaded onto 8% SDS-PAGE gels.
Polypeptides were resolved at 100 V on 8% to 12% gels and
electrophoretically transferred to 0.2-µm nitrocellulose membranes (Schleicher & Schuell Inc) for 1 hour at 100 V. Membranes were blocked
for 1 hour a TBS-T buffer (25 mmol/L Tris, pH 8.0, 150 mmol/L NaCl, and
0.05% Tween-20) containing 3% (wt/vol) nonfat dried milk. Blots were
then probed overnight with primary antibody and developed using a
horseradish peroxidase-coupled anti-mouse secondary antibody by
enhanced chemiluminescence (Supersignal; Pierce Chemical Co, Rockford,
IL) according to the manufacturer's instructions.
Electrophoretic mobility shift (EMSA) assays.
Isolated nuclei were prepared by lysis with Triton X-100 and
centrifugation through a glycerol cushion as described
previously.23 Nuclear protein was extracted using a high
salt, detergent-free buffer containing 20 mmol/L HEPES (pH 7.9), 400 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L EGTA, 1 mmol/L dithiothreitol, and
1 mmol/L phenylmethylsulfonyl fluoride, for at least 20 minutes on ice. Extracts were centrifuged at 4°C for 5 minutes at 12,000g,
and protein content in supernatants was measured by the Bradford
method. A consensus double-stranded NFB probe (obtained from Promega Inc, Madison, WI) was end-labeled using T4 polynucleotide kinase and
 -32P-ATP. Five to 20 µg of nuclear extract was then
incubated in binding buffer (supplied by the manufacturer) containing 1 µg/mL poly dI:dC (Promega), ensuring that the final salt
concentration was between 50 and 100 mmol/L. Reactions were incubated
for 20 minutes at room temperature, and 1 µL of end-labeled probe was added. Samples were incubated for 30 minutes before addition of loading
buffer (Promega) and electrophoresis on 4% nondenaturing polyacrylamide gels that were prerun in 0.5×Tris-borate-EDTA
(TBE) buffer for 30 minutes at 100 V before use. Gels were run at 100 V
in 0.5× TBE and dried, and DNA-protein complexes were detected by
autoradiography.
Statistical analyses.
Mean values and standard deviations were calculated with Microsoft
Excel (Microsoft Inc, Redmond, WA). Significance was
evaluated using two-tailed paired Student's t-tests with SPSS
software (SPSS Inc, Chicago, IL).
| |
RESULTS |
Effects of proteasome inhibitor on DNA fragmentation and surface
exposure of phosphatidylserine.
In a previous study we showed that a specific inhibitor of the NS
protease completely suppressed apoptosis-associated DNA fragmentation
in CLL cells treated with glucocorticoid or the nucleoside analog,
fludarabine.9 Because proteasome inhibitors block apoptosis
in thymocytes and neuronal cells,12,13 and another group
has reported that the NS protease is homologous to the
proteasome,10 we tested the effects of proteasome
inhibitors on DNA fragmentation, measured by PI staining and FACS
analysis, in CLL cells to determine whether the effects of zAPFcmk
might be attributed to the proteasome. Levels were compared with those observed in response to treatment with glucocorticoid hormone. Our
patient isolates fell into three general catetories: (1) those exhibiting relatively high (mean = 40%, n = 10) levels of apoptosis upon in vitro culture in the absence of hormone ("spontaneous"); (2) those exhibiting low spontaneous apoptosis but strong (mean = 40%,
n = 28) increases in DNA fragmentation in response to glucocorticoid treatment ("sensitive"); and (3) those exhibiting low spontaneous apoptosis and low levels of glucocorticoid-induced DNA fragmentation (mean = 10%, n = 21) ("resistant"; Table 1).
Strikingly, and contrary to our expectations, treatment with MG-132, a
peptide-based proteasome antagonist, promoted high levels of DNA
fragmentation in all three categories of cells (Table 1). Proteasome
inhibitors were effective in all patient isolates analyzed (n = 59).
Similar results were obtained with another, structurally distinct
proteasome inhibitor, lactacystin (data not shown). Proteasome
inhibitors also induced surface phosphatidylserine exposure, another
downstream event in apoptosis that is thought to be independent of
endonuclease activation (Fig 1B). Importantly,
preincubation with zAPFcmk blocked MG-132-induced DNA fragmentation
(Fig 1A), indicating that these inhibitors exert their effects on
different biochemical activities.
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| Fig 1.
Induction of apoptosis by proteasome inhibition in CLL
patient isolates. (A) DNA fragmentation analysis. Cells from a
representative glucocorticoid-sensitive patient (see Table 1) were
preincubated in the presence of 25 µmol/L zAPFcmk or 200 µmol/L
zVADfmk for 1 hour and then treated with 10 µmol/L MG132, and DNA
fragmentation was measured at 16 hours by PI staining and FACS
analysis. (B) Surface phosphatidylserine exposure. Cells were incubated
in absence or presence of 10 µmol/L methylprednisolone or 10 µmol/L
MG132 with or without 200 µmol/L zVADfmk, and surface PS exposure was
quantitated by staining with annexin-FITC and measured by FACS
analysis. Results characteristic of three independent experiments with
different patient isolates.
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Effects of MG-132 on DNA fragmentation in normal hematopoietic cells.
In a preliminary attempt to determine whether MG-132's proapoptotic
effects were selective for CLL cells, we analyzed the effects of the
proteasome antagonist on DNA fragmentation in normal peripheral blood
lymphocytes and in G-CSF-mobilized CD34+CD45+
hematopoietic progenitor cells. Normal lymphocytes were killed by the
compound, but the kinetics of the response were markedly delayed and
the maximal response shifted from 16 hours to 48 hours (Fig 2A). Surface staining with specific B- and T-cell
markers indicated that MG-132 was substantially more toxic to normal T cells than to B cells (data not shown). In contrast to the attenuated responses of lymphocytes, mobilized stem cells were highly sensitive to
MG-132 (Fig 2B). Thus, proteasome inhibitors are capable of inducing
apoptosis in certain normal as well as transformed hematopoietic cells.
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| Fig 2.
Effect of proteasome inhibitors on normal lymphocytes.
(A) Effects on normal lymphocytes. Isolated peripheral blood
lymphocytes from normal donors were incubated in the presence of 10 µmol/L MG132 or 10 µmol/L methylprednisolone with or without 25 µmol/L zAPFcmk for 16 hours, and apoptosis was assessed by PI
staining and FACS analysis. Results of one experiment are
representative of three independent replicates. (B) Effects on
hematopoietic progenitor cells. G-CSF-mobilized
CD34+CD45+ progenitor cells were incubated
in the absence or presence of 25 µmol/L zAPFcmk or 10 µmol/L MG-132
and DNA fragmentation was measured after 16 hours by PI staining and
FACS analysis.
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Effects of proteasome inhibition on caspase activation.
Caspases are a family of cysteine proteases that are thought to act at
the core of the apoptotic pathway. Our previous work9 and
that of others24,25 has confirmed that caspases are
required for drug-induced apoptosis in CLL cells. We therefore
investigated whether or not caspases were also required for apoptosis
induced by proteasome inhibitors by four independent approaches. First, induction of apoptosis by proteasome inhibitors was also associated with specific cleavage of the caspase substrate, PARP, as detected by
immunoblotting (Fig 3A). This occurred in all patient
samples analyzed (n = 4), including one that did not respond to
glucocorticoid treatment (Fig 3A). Second, proteasome inhibitors
promoted hydrolysis of a specific caspase substrate (DEVD-AMC; Fig 3B).
Third, proteasome inhibitors induced proteolytic processing of the
inactive form of caspase-3 (procaspase-3; Fig 3C), providing more
direct evidence for activation of caspase-3 and presumably other
caspases in the response. Finally, the caspase inhibitor zVADfmk
completely blocked MG-132-induced DNA fragmentation (Fig 1A) and
surface exposure of phosphatidylserine (Fig 1B), confirming that
caspase activation was required for proteasome inhibitor-induced
apoptosis.
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| Fig 3.
Caspase activation by proteasome inhibitors. (A) Cleavage
of the caspase substrate, PARP, by treatment with proteasome inhibitor.
Cells were treated with either 10 µmol/L methylprednisolone, 25 µmol/L zAPFcmk, or various doses of MG132. PARP was detected by
immunoblotting. Intact (p116) and fragmented (p85) forms of PARP are
indicated by arrows. (B) Effect of MG132 on DEVDase activity. Cells
were treated in the absence or presence of 10 µmol/L MG132 for 8 hours, and hydrolysis of the caspase substrate DEVD-AMC was measured in
a spectrofluorimeter. Results are from two experiments with independent
patient isolates. Cells treated with 10 µmol/L BDcmk, a caspase
inhibitor, did not show DEVDase activity more than 50 U above baseline
levels. DNA fragmentation from these patients was measured in parallel.
These patients are not included in Table 1. Patient no. 1: control = 18.0, MG132 = 96.9; patient no. 2: control = 1.4, MG132 = 32.5. (C) Activation of caspase-3 by proteasome inhibitor. Cells were treated
with either 25 µmol/L zAPFcmk, 10 µmol/L MG132, or 10 µmol/L
methylprednisolone for 16 hours and procaspase-3 was detected by
immunoblotting. Results of one experiment representative of three
replicates with independent patient isolates.
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Effects of proteasome inhibitor on mitochondrial function.
Disruption of mitochondria leading to the release of the electron
transport chain intermediate, cytochrome c, has recently been
implicated in caspase activation in other model systems.26 Although the mechanisms underlying cytochrome c release are unclear, the event is associated with a drop in transmembrane potential (), which may facilitate the opening of transmembrane pores in
the mitochondrial membrane that would allow passage of cytochrome c and
other proapoptotic factors from the organelle.27 To
determine whether this pathway of caspase activation was induced by
proteasome inhibitors in CLL cells, we measured the effects of MG-132
on cytochrome c release in digitonin-permeabilized cells. MG-132 promoted rapid release of cytochrome c from mitochondria in intact CLL
cells (Fig 4, lane 3), effects that were also observed
in cells treated with zAPFcmk (Fig 4, lane 4) and to a lesser extent with glucocorticoid (Fig 4, lane 5). At this time point, control cells
did not exhibit either increased levels of cytosolic cytochrome c (Fig
4, compare lanes 1 and 2) or significant caspase activation (Fig 3B).
The effects of zAPFcmk on cytochrome c release are consistent with
earlier experments that showed that NS protease inhibition results in
caspase activation9 (Fig 3). Proteasome inhibition also
resulted in a drop in mitochondrial membrane potential, measured with the potential-sensitive dye, JC-1
(Fig 5). These results indicate that proteasome inhibitors promote caspase activation via
direct or indirect effects on mitochondria.
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| Fig 4.
Proteasome inhibition leads to release of cytochrome c
from mitochondria. Cells were incubated in the absence (control)
or presence of 10 µmol/L MG132, 25 µmol/L zAPFcmk, or 10 µmol/L
methylprednisolone for 6 hours, and cytosolic cytochrome c was
measured in digitonin-permeabilized cells by immunoblotting. Lane 1, 0 hours control; lane 2, 6 hours control; lane 3, MG132; lane 4, zAPFcmk; lane 5, methylprednisolone. Results are typical of
three independent experiments with different CLL isolates.
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| Fig 5.
Effects of proteasome inhibition on mitochondrial
membrane potential. Cells were incubated in the absence or presence of
10 µmol/L methylprednisolone with or without 25 µmol/L zAPFcmk or
10 µmol/L MG132. Mitochondrial membrane potential was assessed by the
potential sensitive fluorochrome JC-1 and quantitated by FACS
analysis.
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The proteasome is known to degrade many proteins implicated in the
control of cell survival, including p53,28
Fos,29 Jun,30 Myc,31
p27,32 and IB 33 (a protein inhibitor of
the transcription factor NFB34). We did not observe any
detectable alteration in the levels of p53, Fos, Jun, Myc, or p27 in
CLL cells treated with MG-132 compared with cells incubated in medium
alone (data not shown), suggesting that these polypeptides may not
participate in proteasome inhibitor-induced apoptosis in this system.
On the other hand, quantification of NFB activity by EMSA revealed
that both zAPFcmk and MG-132 drastically reduced the levels of active NFB in isolated nuclei from CLL cells
(Fig 6). Thus, blockade of the NFB
survival pathway may be responsible for triggering the disruption of
mitochondrial function and caspase activation in these cells.
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| Fig 6.
Inhibition of NFB activity by NS protease and
proteasome inhibitors. Cells were incubated for 6 hours in the absence
(control) or presence of 25 µmol/L zAPFcmk or 10 µmol/L MG132, and
NFB activity was measured in isolated nuclear extracts by EMSA using
an NFB consensus element DNA probe. Lane 1, control extracts with
excess unlabeled probe (specificity control); lane 2, 6 hours control;
lane 3, zAPFcmk; lane 4, MG-132. Results of one experiment typical of
over 20 independent replicates.
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| |
DISCUSSION |
In spite of the development of nucleoside analogs (fludarabine and
cladribine) that have led to much better management of disease burden
in CLL patients, CLL cells ultimately develop resistance to all
currently available therapies, possibly because of apoptosis suppression. Our data show that the proteasome controls a central step
in the maintenance of cell survival in CLL cells, such that inhibitors
are capable of inducing apoptosis in all of them. The results support
and extend independent work recently published by another group, who
reported that the proteasome inhibitor lactacystin can promote
radiation- and tumor necrosis factor (TNF)-induced apoptosis in CLL
cells.35 The mechanism underlying the responses involves
mitochondrial alterations leading to the release of cytochrome c and
loss of mitochondrial membrane potential. The mitochondrial alterations
are associated with caspase protease activation, as measured by
specific cleavage of hallmark endogenous (PARP) and exogenous
(DEVD-AMC) caspase substrates and proteolytic processing of
procaspase-3. It is encouraging that we were not able to identify a
single patient isolate exhibiting de novo resistance to proteasome inhibition among a fairly large (n = 59) panel, some of which (n = 21)
showed marked resistance to glucocorticoid-induced apoptosis. However,
our work does not address the issue of whether or not CLL cells can
develop resistance to these agents under other conditions. Elimination
of proteasome function in yeast results in lethality,36 but
recent work suggests that mammalian cells contain another protease
complex that can compensate for loss of proteasome function in cells
chronically exposed to proteasome inhibitors.37
Although the precise mechanism(s) precipitating the mitochondrial
changes await further investigation, our preliminary efforts indicate
that the effects of MG-132 are tightly linked to suppression of NFB
activity and not to stabilization of several other proteasome-regulated factors (p53, Fos, Jun, p27) that have been implicated in the control
of apoptosis in other systems. Independent results obtained recently by
another group support the idea that suppression of NFB leads to
apoptosis in CLL.35 The principal mechanism regulating NFB activation involves an inhibitor protein, IB, that binds to NFB and prevents its translocation to the nucleus.38
Stimulation of cells with NFB-activating signals results in
phosphorylation of IB and its coupling to
ubiquitin,33 a small (8 kD) polypeptide that forms polymers
that serve to target proteins for destruction by the
proteasome.39 In other B-cell model systems, constitutive NFB activity is essential for cell survival40 and
inhibition of NFB using protease inhibitors or mutant forms of
IB that cannot be degraded by the proteasome facilitates
apoptosis in a number of different cell types.15-17
Interestingly, the immunosuppressive effects of glucocorticoid hormones
are linked to an inhibition of NFB activity,41-44
suggesting that suppression of NFB may also be required for
glucocorticoid-induced apoptosis. Our ongoing efforts are focused on
further characterizing the role of NFB in the maintenance of
survival in CLL.
Even though some studies had shown that proteasome inhibitors trigger
apoptosis in tumor cell lines,45,46 the observation that
proteasome inhibition results in apoptosis in CLL was surprising to us.
Proteasome inhibitors block glucocorticoid-induced apoptosis in
immature thymocytes12 and the death of neuronal cells
deprived of neurotrophins,13 and ubiquitin-dependent
pathways appear involved in developmentally regulated cell death in the
hawkmoth Manduca sexta and in radiation-induced apoptosis in
tumor cells.47 Furthermore, we had shown that inhibitors of
the NS protease, a putative proteasome homolog, completely block DNA
fragmentation in CLL isolates.9 However, NS protease
inhibitors did promote several other features of apoptosis, including
caspase activation, mitochondrial dysregulation, and PS exposure,
suggesting that their effects did overlap.9 Interestingly,
a peptide inhibitor of the protease complex that can compensate for
loss of proteasome function (AAFcmk)37 is very similar in
sequence to our NS protease inhibitor (APFcmk), both of which contain a
phenylalanine (F) residue at the critical P1 position. In addition,
although MG132 failed to promote substantial accumulation of p53 in CLL
cells, zAPFcmk consistently did (D.J. McConkey, unpublished
observations, April 1998), and the effects of the
inhibitors on NFB activity are similar (Fig 6). We are currently
isolating the NS protease complex, and a detailed analysis of its
structure and biochemical regulation will reveal how it is related (if
at all) to the proteasome.
The proteasome is central to normal cell physiology and hence complete
inhibition of its activity is ultimately cytotoxic. However,
appropriate titration of proteasome activity can elicit significant
efficacy with limited side effects (Peter Elliot, Julian Adams,
Proscript Inc, personal communication, April 1998). Indeed, the present report shows that proteasome inhibitors induce marked apoptosis in mobilized hematopoietic progenitor cells with more
modest effects on normal lymphocytes. Moreover, extensive preclinical
profiling of such compounds has clearly shown that maximum tolerated
doses have only modest myelosuppressive activity (P. Elliot, J. Adams,
personal communication). It is likely that progenitor cell mobilization
and probably other manipulations that induce cell cycling will in
general sensitize cells to proteasome inhibitor-induced apoptosis, as
previous work suggests that proliferating cells are more sensitive to
their effects than are postmitotic cells.48 Additionally,
the proteasome inhibitor, PS-341, has been shown to possess antitumor
activity of its own and this effect is enhanced when combined with
other chemotherapeutics (P. Elliot, J. Adams, personal communication).
With the advent of acceptable phase I safety data, the present results
would strongly argue that PS-341 should be evaluated in patients with
refractory CLL.
| |
ACKNOWLEDGMENT |
The authors thank Virginia Snell for providing the purified
hematopoietic stem cells, Yuko Miyamoto for purified peripheral lymphocytes, and Julian Adams and Peter Elliot (Proscript Inc, Cambridge, MA) for sharing preliminary data on PS-341.
| |
FOOTNOTES |
Submitted February 6, 1998;
accepted July 22, 1998.
Supported by grants from the National Institutes of Health (CA16672,
CA55164, CA49639) (to M.A.), Physicians' Referral Service, MDACC, the
American Cancer Society (RPG-97-169-01-CDD) (to D.J.M.), and an
American Legion Auxiliary Fellowship (to J.C.).
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to David J. McConkey, PhD, Department of Cell
Biology - 173, U.T. M.D. Anderson Cancer Center, 1515 Holcombe Blvd,
Houston, TX 77030; email: dmcconke{at}notes.mdacc.tmc.edu.
| |
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Abstract
Introduction